Coagulation proteases, in addition to their role in the regulation of blood coagulation, can modulate intracellular signaling events by activating a subfamily of G-protein coupled receptors named protease-activated receptors (PARs) expressed on the cell surface of various organs. In vitro studies have indicated that while the anticoagulant protease, activated protein C (APC), in complex with endothelial protein C receptor (EPCR) elicits a barrier protective response via activation of PAR-1 in vascular endothelial cells, thrombin elicits a barrier disruptive response via the activation of the same receptor. APC has been approved by the FDA for treatment of severe sepsis. It has been hypothesized that the activation of PAR-1 by APC on endothelial cells is responsible, at least partially, for its protective properties in preventing organ failure (i.e., lung and heart) which is caused by septic shock in severe sepsis. Thus, it is highly important to understand how the activation of PAR-1 by either APC or thrombin elicits paradoxical protective and disruptive cellular responses, respectively. We recently discovered that the activation of PAR-1 by thrombin can also elicit barrier protective cellular responses if EPCR is occupied by the Gla-domain of protein C. We demonstrated that the unoccupied EPCR together with PAR-1 are both associated with caveolin-1 within lipid-rafts of endothelial cells. Under these conditions, PAR-1 appears to signal via Gq and/or G12/13 subfamily of G-proteins, and thus thrombin activation of PAR-1 leads to disruptive cellular responses. However, we discovered that the occupancy of EPCR by protein C results in dissociation of EPCR from caveolin-1, thereby recruiting PAR-1 to protective pathways by likely coupling it to Gi-protein. Based on our recent preliminary data, we hypothesize that the occupancy of EPCR by protein C can also change the signaling specificity of PAR-2. Thus, it appears that the cell surface occupancy of EPCR determines the signaling specificity of activated PAR-1 and PAR-2 not the proteases that are cleaving them. To understand the PAR-dependent signaling specificity of coagulation proteases, we have prepared several mutant proteases and receptors and set up appropriate in vitro and in vivo models to investigate the following four Specific Aims. In Aim 1, we will investigate the PAR-1- and PAR- 2-dependent signaling specificity of coagulation proteases under conditions in which endothelial cells have been pre-incubated with vitamin K-dependent coagulation zymogens so that to occupy EPCR or other potential receptors by their natural ligands. In Aim 2, we will investigate the contribution of the Gla-domain of vitamin Kdependent coagulation proteases to the recognition and cleavage specificity of PARs localized into the lipidrafts. In Aim 3, we will develop novel APC variants which have improved PAR-1-dependent protective signaling properties, but have reduced anticoagulant activities. In Aim 4, we will characterize the cardioproperties of the improved APC variants or variants possessing only signaling activity or only anticoagulant activity in a mouse ischemia/reperfusion injury model. PUBLIC HEALTH RELEVANCE: The studies of this application will focus on the mechanism by which coagulation proteases activate a subfamily of G-protein coupled receptors called protease-activated receptors to alter the properties of vascular endothelial cells. The activation of these receptors by coagulation proteases is known to be important for the regulation of a range of (patho) physiological processes including coagulation, inflammation and angiogenesis. Understanding this role of coagulation proteases can lead to development of a new generation of therapeutic drugs for controlling thrombotic and inflammatory disorders, including heart attack and severe sepsis.